Aedes aegypti is the principal vector of dengue in urban areas. Despite its epidemiological importance, the genetic variability of Colombian populations of this species is unknown.To determine the genetic variability of mitocondrial gene ND4, which codes for subunit 4 of the enzyme NADH deshydrogenase, between populations of Ae. aegypti from municipalities of Sincelejo and Guaranda. The incidences of dengue reported from these two localities are high and low, respectively.Genetic material extracted from 36 females of Ae. aegypti was used to determine the partial sequence of the mitocondrial gene ND4 as well as to estimate the parameters of nucleotidic and haplotypic diversities, genetic structure and gene flow between the Sincelejo and Guaranda populations. The molecular variance was also analysed and a haplotypic network constructed.In all 36 nucleotide sequences of 282 pb were obtained. These presented 12 polymorphic sites and could grouped into 10 haplotypes, two of them present in both populations, three exclusive to the Sincelejo population and five to that of Guaranda. The estimators of genetic structure (FST = 0.15) and gene flow (Nm = 1.40) are both indicative of genetic differentiation and a limited exchange of genes between the populations.The Sincelejo and Guaranda populations of Ae. aegypti are genetically divergent.